Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing gamma herpesvirus, which establishes life-long infection in humans. KSHV accounts for the development of Kaposi's sarcoma, primary effusion lymphoma (PEL), and aggressive forms of multicentric Castleman's disease in immunocompromised patients. Life cycle of KSHV has two distinct phases: latency and lytic replication. While latent infection has been traditionally linked to KSHV tumorigenesis, accumulating evidence suggests that lytic replication of KSHV is important for not only the spreading of the virus but also KSHV-associated oncogenesis. The lytic cycle of KSHV is initiated by the expression of the replication and transcription activator (RTA) encoded by KSHV. RTA is required and sufficient for inducing lytic reactivation of KSHV from latently infected cells. Despite the essential role of RTA, it is still poorly understood how RTA can manage to rapidly induce a large variety of its target genes on the highly chromatinized viral and host genomes that are required for lytic replication of KSHV. To uncover the mechanisms of RTA-mediated gene regulation in KSHV-infected cells, we have been studying the transcriptional function of RTA in PEL cells undergoing reactivation. We performed ChIP-seq analyses to determine the binding of RTA and the enrichment of activating histone modifications on both viral and host genomes during latency and lytic reactivation. RNA-seq was also used to analyze the differential gene expression of viral and host genes between latency and lytic reactivation in PEL cells. These analyses revealed the direct gene targets of RTA and the chromatin changes associated with RTA-binding. The functional study of an RTA mutant showed that RTA can play a role in the regulation of RNA polymerase II elongation. In agreement with this, RTA complex purification using reactivated PEL cells showed that RTA interacts with several viral factors associated with DNA replication and transcription, host transcription elongation factors, chromatin modifying enzymes, and nucleosome remodeling factors. Based on our preliminary results, we hypothesize that RTA utilizes the chromatin regulatory and transcription elongation host factors in concert with viral co-factors to induce its viral and host target genes for promoting KSHV reactivation from latency and induce viral replication. Collectively, the goal of this proposal is to determine the genome-wide effect of RTA on the regulation of transcription elongation on both the viral and host genomes (Aim 1), and dissect the mechanisms of how RTA utilizes the chromatin modifying and remodeling host factors, the transcription elongation host factors, and viral co-factors to facilitate the progression of RNA polymerase II at RTA target genes during lytic reactivation of KSHV (Aim 2). This is a highly innovative proposal, which combines genomics with biochemical assays to help better understand the molecular mechanisms of lytic reactivation from latently infected B cells, which has biological importance in KSHV pathogenesis.